ABSTRACT
To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer novel insights into the physiology of production and role of saliva, blood digestion, energy metabolism, and development with submission of 10,932 novel tissue/cell specific CDS to the NCBI database for this important tick species.
Subject(s)
Gene Expression Profiling/methods , Gene Regulatory Networks , Rhipicephalus/physiology , Animals , Cattle , Female , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Organ Specificity , Ovary/chemistry , Pregnancy , Rhipicephalus/genetics , Saliva/chemistry , Sequence Analysis, RNAABSTRACT
In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.
Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Trypanosoma brucei brucei/enzymology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/drug effects , Animals , Calcium Chloride/pharmacology , Chlorides/pharmacology , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , Hydrolysis , Magnesium Chloride/pharmacology , Manganese Compounds/pharmacology , Suramin/pharmacology , Time Factors , Trypanocidal Agents/pharmacology , Zinc Compounds/pharmacologyABSTRACT
Free D-amino acids are implicated in several biological functions. This study examined the presence of D-alanine in Leishmania amazonensis. Measuring chiral amino acid content by high-performance liquid chromatography we detected a significant amount of free D-alanine in promastigotes of these parasites. D-alanine accounts for 8.9% of total free alanine and is found primarily in the soluble fraction. Specific racemization of L-alanine to D-alanine was detected in cell lysates and this enzyme activity was inhibited by D-cycloserine, an alanine racemase inhibitor. Furthermore, we were able to decrease this pool of D-amino acid by treating our cultures with D-cycloserine. We demonstrate for the first time the existence of a significant amount of free D-alanine in L. amazonensis and an alanine racemase activity present in cell lysates. The restriction of D-alanine to bacteria, some fungi and now in L. amazonensis opens a new perspective on treatment of diseases caused by these microorganisms.
